Vsig8-based chimeric proteins

Abstract

The present invention relates, in part, to, chimeric proteins which include the extracellular domain of V-set and immunoglobulin domain-containing protein 8 (VSIG8) and their use in the treatment of diseases, such as immunotherapies for cancer and/or inflammatory diseases.

What is claimed is: 
     
         1 . A heterologous chimeric protein comprising:
 (a) a first domain comprising a portion of V-set and immunoglobulin domain-containing protein 8 (VSIG8) that is capable of binding a VSIG8 ligand;   (b) a second domain comprising a portion of OX40 ligand (OX40L) that is capable of binding a OX40L receptor; and   (c) a linker linking the first domain and the second domain.   
     
     
         2 . The heterologous chimeric protein of  claim 1 , wherein the first domain comprises substantially all of the extracellular domain of VSIG8 and the second domain comprises substantially all of the extracellular domain of OX40L. 
     
     
         3 . The heterologous chimeric protein of  claim 1  or  claim 2 , wherein the heterologous chimeric protein is capable of inhibiting an immunosuppressive signal. 
     
     
         4 . The heterologous chimeric protein of  claim 1  or  claim 2 , wherein the heterologous chimeric protein is capable of:
 (a) reducing, disrupting, or eliminating an immune inhibitory signal when the portion of VSIG8 is bound to its ligand and/or 
 (b) increasing, stimulating, or activating an immune stimulatory signal when the portion of OX40L is bound to its receptor. 
 
     
     
         5 . The heterologous chimeric protein of any one of  claims 1  to  4 , wherein the VSIG8 ligand is V-region Immunoglobulin-containing Suppressor of T cell Activation (VISTA). 
     
     
         6 . The heterologous chimeric protein of any one of  claims 1  to  5 , wherein the OX40L receptor is OX40. 
     
     
         7 . The heterologous chimeric protein of any one of  claims 1  to  6 , wherein the heterologous chimeric protein is capable of contemporaneously binding the VSIG8 ligand and the OX40L receptor, wherein the VSIG8 ligand is VISTA and the OX40L receptor is OX40. 
     
     
         8 . The heterologous chimeric protein of any one of  claims 1  to  7 , wherein the chimeric protein is capable of contemporaneously binding recombinant human OX40 and human VISTA in vitro. 
     
     
         9 . The heterologous chimeric protein of any one of  claims 1  to  8 , wherein the linker is a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence. 
     
     
         10 . The heterologous chimeric protein of  claim 9 , wherein the linker comprises hinge-CH2-CH3 Fc domain derived from IgG4. 
     
     
         11 . The heterologous chimeric protein of  claim 10 , wherein the hinge-CH2-CH3 Fc domain is derived from human IgG4. 
     
     
         12 . The heterologous chimeric protein of any one of  claims 1  to  11 , wherein the chimeric protein is expressed by a mammalian host call as a secretable and functional single polypeptide chain. 
     
     
         13 . The heterologous chimeric protein of any one of  claims 1  to  12 , wherein the portion of VSIG8 is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         14 . The heterologous chimeric protein of any one of  claims 1  to  13 , wherein the portion of OX40L is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. 
     
     
         15 . The heterologous chimeric protein of any one of  claims 1  to  14 , wherein the linker comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. 
     
     
         16 . The heterologous chimeric protein of any one of  claims 1  to  15 , wherein the linker comprises one or more joining linkers, such joining linkers independently selected from SEQ ID NOs: 28 to 74. 
     
     
         17 . The heterologous chimeric protein of  claim 16 , wherein the linker comprises two or more joining linkers each joining linker independently selected from SEQ ID NOs: 28 to 74; wherein one joining linker is N terminal to the hinge-CH2-CH3 Fc domain and another joining linker is C terminal to the hinge-CH2-CH3 Fc domain. 
     
     
         18 . The heterologous chimeric protein of any one of  claims 1  to  17 , wherein the chimeric protein is a recombinant fusion protein. 
     
     
         19 . The heterologous chimeric protein of any one of  claims 1  to  18 , wherein the chimeric protein enhances the recognition of tumor antigen by CD8+ T cells. 
     
     
         20 . The heterologous chimeric protein of any one of  claims 1  to  19 , wherein the chimeric protein depletes VISTA, optionally in the serum. 
     
     
         21 . The heterologous chimeric protein of any one of  claims 1  to  20 , wherein the chimeric protein exhibits enhanced anti-tumor effects compared to OX40 agonist antibodies and/or VSIG8 antagonistic antibodies. 
     
     
         22 . The heterologous chimeric protein of any one of  claims 1  to  21 , wherein the heterologous chimeric protein is capable of increasing or preventing a decrease in a sub-population of CD4+ and/or CD8+ T cells. 
     
     
         23 . The heterologous chimeric protein of any one of  claims 1  to  22 , wherein the heterologous chimeric protein is capable of enhancing tumor killing activity by T cells. 
     
     
         24 . The heterologous chimeric protein of any one of  claims 1  to  23 , wherein the heterologous chimeric protein is capable of providing a sustained immunomodulatory effect. 
     
     
         25 . The heterologous chimeric protein of any one of  claims 1  to  24 , wherein the heterologous chimeric protein is capable of causing activation of antigen presenting cells. 
     
     
         26 . The heterologous chimeric protein of any one of  claims 1  to  25 , wherein the heterologous chimeric protein is capable enhancing the ability of antigen presenting cells to present antigen. 
     
     
         27 . The heterologous chimeric protein of any one of  claims 1  to  26 , wherein the chimeric protein shifts the ratio of immune cells in favor of cells that can kill a tumor in opposition to cells that protect tumors. 
     
     
         28 . The heterologous chimeric protein of any one of  claims 1  to  27 , wherein the chimeric protein increases the ratio of effector T cells to regulatory T cells. 
     
     
         29 . The heterologous chimeric protein of any one of  claims 1  to  28 , wherein the chimeric protein provides a sustained masking effect of immune inhibitory signals. 
     
     
         30 . The heterologous chimeric protein of any one of  claims 1  to  29 , wherein the chimeric protein provides longer on-target (e.g., intra-tumoral) half-life (t 1/2 ) as compared to serum tin of the chimeric proteins. 
     
     
         31 . The heterologous chimeric protein of any one of  claims 1  to  30 , wherein the chimeric protein reduces toxicities as compared with treatment with antibodies against VSIG8 and OX40. 
     
     
         32 . The heterologous chimeric protein of any one of  claims 1  to  31 , wherein the chimeric protein is capable of forming a stable synapse between cells. 
     
     
         33 . The heterologous chimeric protein of  claim 32 , wherein the stable synapse between cells provides spatial orientation that favors tumor reduction. 
     
     
         34 . The heterologous chimeric protein of  claim 32  or  claim 33 , wherein the spatial orientation positions T cells to attack tumor cells and/or sterically prevents a tumor cell from delivering negative signals, including negative signals beyond those masked by the chimeric protein of the invention. 
     
     
         35 . The heterologous chimeric protein of any one of  claims 1  to  34 , wherein binding of either or both of the extracellular domains to its respective binding partner occurs with slow off rates (Koff), which provides a long interaction of a receptor and its ligand. 
     
     
         36 . The heterologous chimeric protein of  claim 35 , wherein the long interaction delivers a longer positive signal effect. 
     
     
         37 . The heterologous chimeric protein of  claim 36 , wherein the longer positive signal effect allows an effector cell to be adequately stimulated for an anti-tumor effect. 
     
     
         38 . The heterologous chimeric protein of  claim 36  or  claim 37 , wherein the long interaction provides T cell proliferation and allows for anti-tumor attack. 
     
     
         39 . The heterologous chimeric protein of any one of  claims 36  to  38 , wherein the long interaction allows sufficient signal transmission to provide release of stimulatory signals. 
     
     
         40 . The heterologous chimeric protein of  claim 39 , wherein the stimulatory signal is a cytokine. 
     
     
         41 . An expression vector, comprising a nucleic acid encoding the heterologous chimeric protein of any one of  claims 1  to  40 . 
     
     
         42 . A host cell, comprising the expression vector of  claim 41 . 
     
     
         43 . A pharmaceutical composition, comprising a therapeutically effective amount of the heterologous chimeric protein of any one of  claims 1  to  40 . 
     
     
         44 . A method of treating cancer or an inflammatory disease, comprising administering an effective amount of a pharmaceutical composition of  claim 43  to a subject in need thereof. 
     
     
         45 . A method of modulating a patient's immune response, comprising administering an effective amount of a pharmaceutical composition of  claim 43  to a subject in need thereof. 
     
     
         46 . The method of  claim 44  or  claim 45 , wherein the patient's T cells are activated. 
     
     
         47 . The method of  claim 44  or  claim 45 , wherein the patient has a tumor and one or more tumor cells are prevented from transmitting an immunosuppressive signal. 
     
     
         48 . A method for treating cancer or an inflammatory disease comprising administering an effective amount of a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising a heterologous chimeric protein comprising:
 (a) a first domain comprising a portion of colony stimulating factor 1 receptor (VSIG8) that is capable of binding a VSIG8 ligand,   (b) a second domain comprising a portion of CD-40 ligand (OX40L) that is capable of binding an OX40L receptor, and   (c) a linker linking the first domain and the second domain.   
     
     
         49 . The method of  claim 48 , wherein the subject's T cells are activated when bound by the second domain of the heterologous chimeric protein and:
 (a) one or more tumor cells are prevented from transmitting an immunosuppressive signal when bound by the first domain of the heterologous chimeric protein,   (b) a quantifiable cytokine response in the peripheral blood of the subject is achieved, and/or   (c) tumor growth is reduced in the subject in need thereof as compared to a subject treated with OX40 blocking antibodies and/or VISTA blocking antibodies.   
     
     
         50 . The method of any one of  claims 44  to  49 , wherein the method stimulates OX40 signaling and activates antigen-presenting cells. 
     
     
         51 . The method of any one of  claims 44  to  50 , wherein the method reduces the amount or activity of regulatory T cells (Tregs) as compared to untreated subjects or subjects treated with agents targeting one of OX40/OX40L and VISTA/VSIG8. 
     
     
         52 . The method of any one of  claims 44  to  51 , wherein the method increases priming of effector T cells in draining lymph nodes of the subject as compared to untreated subjects or subjects treated with agents targeting one of OX40/OX40L and VISTA/VSIG8. 
     
     
         53 . The method of any one of  claims 44  to  52 , wherein the method causes an overall decrease in immunosuppressive cells and a shift toward a more inflammatory tumor environment as compared to untreated subjects or subjects treated with agents targeting one of OX40/OX40L and VISTA/SIG8. 
     
     
         54 . The heterologous chimeric protein of any one of  claims 1  to  40  for use as a medicament. 
     
     
         55 . The heterologous chimeric protein of any one of  claims 1  to  40  for use in the treatment of cancer. 
     
     
         56 . The heterologous chimeric protein of any one of  claims 1  to  40  for use in the treatment of an inflammatory disease. 
     
     
         57 . Use of the chimeric protein of any one of  claims 1  to  40  in the manufacture of a medicament. 
     
     
         58 . A recombinant fusion protein comprising a general structure of:
   N terminus-(a)-(b)-(c)-C terminus,   wherein:
 (a) is a first domain comprising an extracellular domain of VSIG8 that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 and is capable of binding a VSIG8 ligand, 
 (b) is a linker linking the first domain and the second domain and comprising a hinge-CH2-CH3 Fc domain derived from human IgG4, and optionally a joining linker sequence from SEQ ID 28 to 74, and 
 (c) is a second domain comprising an extracellular domain of CD-40 ligand (OX40L) that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and is capable of binding an OX40L receptor. 
   
     
     
         59 . The recombinant fusion protein of  claim 58 , wherein the linker comprises a sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27. 
     
     
         60 . The recombinant fusion protein of  claim 58  or  claim 59 , for use as a medicament. 
     
     
         61 . The recombinant fusion protein of any one of  claims 58  to  60  for use in the treatment of cancer. 
     
     
         62 . The recombinant fusion protein of any one of  claims 58  to  60 , for use in the treatment of an inflammatory disease. 
     
     
         63 . Use of the recombinant fusion protein of any one of  claims 58  to  60 , in the manufacture of a medicament.